Research Project: Innovative Food and Feed Safety Research to Eliminate Mycotoxin Contamination in Corn and other Crops

Location: Mycotoxin Prevention and Applied Microbiology Research

2023 Annual Report

Objectives
Objective 1: Define diversity of mycotoxin-producing Fusarium species. [C1, PS1, PS2] Sub-objective 1.A: Elucidate phylogenetic diversity, mycotoxin potential, and pathogenicity to cereals of fungi in the F. tricinctum species complex. Sub-objective 1.B: Identify genomic and phenotypic differences in collections of F. proliferatum and F. verticillioides isolates to aid discovery of targets for control of fumonisins in corn. Objective 2: Identify targets to reduce fumonisin contamination in corn. [C1, PS1, PS2, PS5] Sub-objective 2.A: Determine whether the corn zmCRR1 protein contributes to resistance to fumonisin contamination. Sub-objective 2.B: Identify corn genes encoding papain-like cysteine proteases involved in fumonisin contamination to aid genomics-assisted breeding. Sub-objective 2.C: Reduce fumonisin contamination in corn by engineering kernel-specific expression of RNAi targeting the fumonisin biosynthetic gene FUM1. Sub-objective 2.D: Determine how corn oxylipins control fumonisin production in F. verticillioides. Sub-objective 2.E: Determine whether the killer meiotic drive element SkK can be used to drive biased transmission of a gene that blocks fumonisin production in F. verticillioides.

Approach
Fusarium species are fungi with potentially the greatest negative impact on agriculture. This is because of their collective abilities to produce mycotoxins and cause destructive diseases in crops, including the important cereals: corn, wheat, and rice. The Fusarium mycotoxins fumonisins and trichothecenes are among the mycotoxins of most concern to food and feed safety due to their toxicity and frequent occurrence in crops. However, Fusarium species produce other mycotoxins whose effects on food and feed safety are poorly understood. In the U.S., harmful impacts of mycotoxins on health are mitigated by removing contaminated grain from food/feed supply chains. Despite these efforts, however, the toxins continue to cause billions of dollars in agricultural losses. This project plan addresses knowledge gaps that hinder control of mycotoxins caused by two groups of Fusarium: the Fusarium tricinctum species complex (FTSC), which includes multiple species that cause head blight of small-grain cereals and produce multiple mycotoxins; and the F. fujikuroi species complex, specifically Fusarium proliferatum and Fusarium verticillioides, which are the primary causes of fumonisin contamination in corn. The proposed research has two objectives: i) define diversity of mycotoxin-producing Fusarium species, specifically members of the FTSC, F. proliferatum, and F. verticillioides; and ii) identify targets to reduce fumonisins in corn. To address the first objective, we propose to elucidate variation in genome sequences, mycotoxin production, and pathogenicity within and among Fusarium species. This will aid development of broadly effective control practices for Fusarium mycotoxins. To address the second objective, we propose to identify corn and Fusarium proteins/genes that can be used to enhance breeding or engineering strategies aimed at reducing fumonisin contamination. To address the second objective, we also propose to develop fumonisin reduction methods based on two biological phenomena: RNA interference and meiotic drive elements. The research accomplishments will aid efforts to reduce mycotoxin contamination in corn and other cereal crops, and will benefit growers, processors, regulatory agencies, and ultimately American consumers.

Progress Report
Objective 1: Fumonisins are fungal toxins that contaminate corn and pose threats to food safety. Although the fumonisin-producing fungi Fusarium proliferatum and Fusarium verticillioides are the main causes of the contamination, it is not clear how strains of the fungi from different crops or geographic regions differ genetically and whether genetic diversity affects control practices to reduce fumonisin contamination. Therefore, to assess genetic diversity, we assembled worldwide collections of approximately 90 isolates of each species, generated genome sequence data of each isolate, and are determining how the sequences differ with in and among the two species. To do this, we developed a series of computer-based analyses to identify sequence differences among isolates of each species. Thus far, the results indicate that F. proliferatum is more genetically diverse than F. verticillioides, which is consistent with the occurrence of the former species on a wider range of crops. Understanding the genetic diversity of fumonisin-producing fungi will aid development of plant breeding and other efforts to reduce fumonisin contamination. Objective 1: Fusarium head blight (FHB) is a devastating fungal disease of wheat and barley that reduces the quality and yield but also leaves grain contaminated with toxins that are a food safety hazard. In collaboration with researchers at North Carolina State University and ARS researchers in Raleigh, North Carolina, we examined the frequency and diversity of fungi known as the Fusarium tricinctum species complex (FTSC) that cause FHB of wheat, including their ability to produce the toxins enniatins. We found that over 80% of the fungal isolates analyzed produced enniatins, which are a concern because their effects on human and animal health are not well understood. The results of this research will aid plant pathologists, plant breeders, and regulatory organizations to assess risks the FTSC poses to food safety. Objective 1: Crop debris left in fields after harvest is a source of inoculum for crop diseases the next year. It is unclear whether the ability to overwinter on this debris differs among toxin-producing species of the fungus Fusarium. In the Midwest, corn is frequently planted into fields where corn was grown the previous year. In such fields, corn seeds are planted into soil with abundant corn debris. Fungicide applications to control corn stem and foliar diseases may have two unintended consequences: 1) the applications could reduce degradation of the debris by reducing the diversity and abundance of fungi involved in decomposition and 2) reduced decomposition could allow more Fusarium species to overwinter in the debris, increasing amount of fungi able to cause disease the following year. Analysis of corn leaves prior to harvest indicated that fungicide applications changed the abundance of debris-decomposing fungi. Experiments are underway to determine whether changes in abundance of these fungi in the leaves one year affects abundance of Fusarium species the next year. Objective 1: A major limitation to corn production is fusarium ear rot (FER), which is caused primarily by the fungus Fusarium verticillioides, which produces the toxins fumonisins. In collaboration with researchers at Addis Ababa and Jimma Universities in Ethiopia, we surveyed 480 agricultural fields in Ethiopia for FER. The survey revealed that FER was widespread, and fields with higher FER severity had lower yield. Insect and weed infestation and low nitrogen levels were associated with high levels of FER. Agricultural practices such as early planting, tillage, crop rotation, and intercropping were associated with lower FER and higher yield. The results also confirmed a positive association between FER severity and fumonisin contamination. The research also revealed that multiple species of Fusarium are associated with FER in Ethiopia. This study led to recommendations of practices to aid Ethiopian farmers manage FER and to reduce fumonisin contamination. Objective 1: Recent predictive models for mycotoxin risk in Illinois showed higher risk of fumonisin mycotoxins in southern counties. Corn samples from each Illinois county collected by the Illinois Department of Agriculture in 2021 and 2022 were used to determine if differences in mycotoxin-producing Fusarium species in corn were associated with differences in fumonisin contamination levels in northern vs southern counties. Mycotoxin-producing Fusarium species were isolated and characterized using morphological and DNA sequence-based methods. Fusarium species identified included F. verticillioides, F. subglutinans, F. andiyazi, F. poae, F. graminearum, F. fujikuroi, and F. proliferatum. F. verticillioides was the most frequently recovered species and occurred at a higher frequency in southern counties than northern counties. Three genetic variants of F. verticillioides were identified and are being examined to determine if they differ in agriculturally important traits, such as growth, ability to cause disease, temperature and carbon dioxide tolerance, and toxin production. Objective 2: Secondary metabolites (SMs) are metabolites that are not essential for growth, but allow organisms to survive, exploit resources, and/or compete with other organisms under certain conditions. Fungal SMs include toxins that pose health hazards to humans and animals. In fungi, genes directly involved in synthesis of a SM tend to be located adjacent to one another in what is known as biosynthetic gene clusters. Understanding how biosynthetic gene clusters are distributed among species of the fungus Fusarium can aid assessments of the risk that individual mycotoxin-producing species pose to food/feed safety. We used computer-based Artificial Intelligence (AI)/Machine Learning (ML) approaches in combination with our database of over 600 Fusarium genome sequences to identify biosynthetic gene clusters and to predict structures and biological activity of SM products of novel gene clusters that we identified. The results of this research are aiding assessments of risks to food/feed safety posed by Fusarium species and are also providing information on potential targets for development of strategies to reduce mycotoxin contamination. Objective 2: In order to identify other targets to reduce fumonisin contamination in corn, we are investigating the complex interaction of plant and fungal proteins that can determine whether fungi cause crop diseases and mycotoxin contamination, or whether plants can defend themselves against fungi and block development of disease and mycotoxin contamination. As part of this, we determined how one group of plant proteins protects another group of plant proteins that inhibit fungi. We also found that the mycotoxin-producing fungi Fusarium verticillioides and Fusarium graminearum produce proteins that degrade the protective plant proteins. We also found that the Fusarium proteins triggers an immune response in corn. Such responses can enhance resistance of corn to fungal diseases. We are also investigating another group of plant proteins, known as papain-like proteases, that trigger plant immune responses. We have found that some plant disease-causing species of Fusarium secrete proteins that specifically inhibit a corn papain-like protease. The identification of corn and Fusarium proteins that interact with one another to affect occurrence of crop diseases will aid development of corn hybrids that are resistance to Fusarium-incited diseases and, in turn, reduce mycotoxin contamination. Objective 2: We are developing a database of three-dimensional (3D) structures of all proteins produced by the mycotoxin-producing Fusarium species F. graminearum and F. verticillioides. Understanding protein 3D structures aids in determining their function and how they interact with other molecules, including other proteins. The database is being used to identify Fusarium proteins that interact with plant proteins, thereby allowing fungi to cause disease and mycotoxin contamination in corn. Knowledge gained from this research will aid development of control strategies to reduce mycotoxin contamination in crops. Objective 2: We conducted experiments to determine how a group of fatty acids known as oxylipins impact production of fumonisin mycotoxins by the fungus Fusarium verticillioides. We discovered that different oxylipins have markedly different effects on expression of hundreds of genes in F. verticillioides, including the genes that are directly involved in fumonisin production. Our results also indicate that the effects of oxylipins on gene expression in F. verticillioides is dependent on the conditions under which the fungus is grown. We are using these findings to elucidate genetic mechanisms that turn fumonisin production on and off in F. verticillioides. Understanding these mechanisms will aid identification of genes that block fumonisin production in the fungus and have potential to prevent fumonisin contamination in corn. Objective 2: We are also conducting research to harness a naturally occurring process called RNA interference (RNAi) to reduce mycotoxin contamination in crops. The process occurs when small RNA molecules suppress expression of a target gene. We are developing an RNAi system to control fumonisin mycotoxin contamination in corn. We contracted with the Wisconsin Crop Innovation Center to create corn plants that produce RNAi molecules that suppress expression of a gene (FUM1) that is required for fumonisin production in the fungus Fusarium verticillioides. The corn plants engineered to produce FUM1-specific RNAi molecules are currently being grown at the Wisconsin center, which will send the resulting seed to ARS researchers in Peoria, Illinois, in the summer of 2023. The plants resulting from these seed will be evaluated for their ability to suppress fumonisin contamination when infected by F. verticillioides.

Accomplishments
1. Controlling mycotoxin by harnessing a naturally occurring mechanism that modifies genetic inheritance. Corn is the most valuable farm crop in the U.S. The fungus Fusarium verticillioides can infect corn and produce a family of toxins called fumonisins that are hazards to human and animal health. Because fumonisins are being detected with increasing frequency in U.S. corn, new control strategies are needed to limit fumonisin contamination and the resulting monetary losses. Therefore, ARS researchers in Peoria, Illinois, and at Illinois State University have identified a gene (SKC1) that blocks the inheritance of other genes when the fungus undergoes sexual reproduction. The researchers also demonstrated that effect of SKC1 is mediated by a naturally occurring genetic process that protects F. verticillioides DNA from viruses and other gene-damaging processes. This improvement to understanding how SKC1 functions has potential to enhance efforts to use this and similar genes to control crop diseases and mycotoxin contamination. Thus, the results of this research will be used by plant breeders, plant genetic engineers, and plant pathologists working to reduce mycotoxin contamination in corn and other crops.

2. Discovery of a fungal protein that degrades a plant defense protein and has potential to enhance efforts to control crop disease. Reducing fungal diseases of crops that reduce yield and contaminate grains with toxins will ensure the safety of the U.S. food supply. Some crop diseases occur when pathogen proteins impair the plant immune system. Characterization of such proteins can be a critical step in efforts to control crop diseases by plant breeding and engineering. ARS researchers in Peoria, Illinois, discovered a protein in the fungal cotton pathogen Verticillium that digests a plant defense protein. The researchers used this information to identify the same protein in Fusarium species that cause disease and mycotoxin contamination in corn, wheat, and barley. This discovery is a critical step in understanding interactions of plant and fungal proteins that result in disease and mycotoxin contamination of crops. Selecting plant proteins that are resistant to the fungal proteins in natural plant populations or by genetic engineering will help plant breeders produce crops with improved resistance to disease and mycotoxin contamination. The improved resistance will, in turn, reduce economic losses for farmers and improve the safety and security of food and feed.

3. Determined structure of a novel fungal enzyme that promotes disease. Fungal diseases of crops cost farmers billions of dollars each year and will increase with increasing global temperatures. Some fungi secrete enzymes that digest plant defense proteins known as chitinases. ARS researchers in Peoria, Illinois, and researchers at the University of Waterloo, Ontario, Canada, determined the 3-D structure of an enzyme called polyglycine hydrolase that degrades plant chitinases. The successful determination of the structure was made possible by combining X-ray technology data with an artificial intelligence program called RoseTTAFold. Knowing the 3-D structures of proteins aids in understanding how they function and interact with other molecules, including other proteins. Understanding how the proteins function will aid identification or genetic engineering of plant chitinases that resist degradation. This in turn will aid development of crop plants with resistance to diseases and mycotoxin contamination. The improved resistance will reduce crop losses and improve safety of food and feed.

4. Generated high-quality genome sequences of fungi to aid development of strategies to control diseases and toxin contamination of crops. Genome sequence data of toxin-producing fungi aids development of strategies to control toxin contamination of crops. However, the utility of most publicly available genome sequences for toxin-producing species of Fusarium is limited due to the moderate quality of the data, which results in genome sequences consisting of fragmented chromosomes. Therefore, researchers at ARS in Peoria, Illinois, the University of Massachusetts, and the U.S. Department of Energy’s Joint Genome Institute (JGI) generated high-quality, data for 18 species of the mycotoxin-producing fungus Fusarium, data that resulted in genome sequences consisting of full-length chromosomes. The data are available to researchers worldwide at JGI’s MycoCosm website. The high-quality genome sequences will serve as references that aid in understanding genetic diversity that enables Fusarium species to cause disease and mycotoxin contamination of crops. Such information is essential to develop robust control strategies that reduce crop diseases and mycotoxin contamination.

5. Sugar biosynthetic enzyme protects Fusarium from stress. Corn is the most widely grown crop in the U.S. and is a staple food around the world. The fungus Fusarium verticillioides infects corn and contaminates kernels with toxins called fumonisins. Plant disease and toxin contamination of corn cause billion-dollar losses world-wide. ARS researchers in Peoria, Illinois, and researchers at Bradley University discovered that the biosynthetic enzyme for the sugar trehalose also functions to protect F. verticillioides from drought stress that is unrelated to trehalose synthesis. Understanding how Fusarium protects itself from stress will aid plant pathologists and other researchers in developing new strategies to reduce toxin contamination of grain to ensure a safe food supply.

6. Discovered new Fusarium species that are potential hazards to food and feed safety. A safe and secure food supply needs new methods to control crop diseases and mycotoxin contamination caused by Fusarium. An important part of this process is knowing which species of Fusarium cause specific problems and distinguishing them from other species. ARS researchers in Peoria, Illinois, analyzed a group of Fusarium species known as the Fusarium buharicum species complex and showed that the complex consists of at least seven species, including four new species. They also determined which mycotoxins each species produces and found that one species produced trichothecene mycotoxins, which pose health hazards to humans and animals. Knowledge of the mycotoxin production abilities of fungi in the F. buharicum species complex aids understanding of the risks that the fungi pose to food and feed safety.

Review Publications
Naumann, T.A., Hao, G., Dowd, P.F., Johnson, E.T., Naldrett, M.J., Price, N.P.J. 2022. Discovery of a secreted Verticillium dahliae protease that cleaves cotton CRR1 and induces plant cell death. Physiological and Molecular Plant Pathology. 123. Article 101941. https://doi.org/10.1016/j.pmpp.2022.101941.
Lohmar, J.M., Rhoades, N.A., Hammond, T.M., Brown, D.W. 2022. Gene drive by Fusarium SKC1 is dependent on its competing allele. Fungal Genetics and Biology. 163. Article 103749. https://doi.org/10.1016/j.fgb.2022.103749.
Dowling, N.V., Naumann, T.A., Price, N.P.J., Rose, D.R. 2023. Crystal structure of a polyglycine hydrolase determined using a RoseTTAFold model. Acta Crystallographica, Section D: Structural Biology. 79:168-176. https://doi.org/10.1107/S2059798323000311.
O'Donnell, K., Grafenhan, T., Laraba, I., Busman, M., Proctor, R.H., Kim, H.-S., Wiederhold, N.P., Geiser, D.M., Seifert, K.A. 2022. Fusarium abutilonis and F. guadeloupense, two novel species in the Fusarium buharicum clade supported by multilocus molecular phylogenetic analyses. Mycologia. 114(4):682-696. https://doi.org/10.1080/00275514.2022.2071563.
Yli-Mattila, T., Opoku, J., Ward, T.J. 2023. Population structure and genetic diversity of Fusarium graminearum from southwestern Russia and the Russian Far East as compared to northern Europe and North America. Mycologia. 115(4):513-523. https://doi.org/10.1080/00275514.2023.2198927.
Vaughn, S.F., Liu, S.X., Berhow, M.A., Moser, J.K., Peterson, S.C., Selling, G.W., Hay, W.T., Jackson, M.A., Skory, C.D. 2023. Production of an odor-reducing, low-dust, clumping cat litter from soybean hulls and soybean hull biochar. Bioresource Technology Reports. 21. Article 101317. https://doi.org/10.1016/j.biteb.2022.101317.
Flor-Weiler, L., Behle, R.W., Berhow, M.A., McCormick, S.P., Vaughn, S.F., Muturi, E.J., Hay, W.T. 2023. Bioactivity of brassica seed meals and its compounds as ecofriendly larvicides against mosquitoes. Scientific Reports. 13. Article 3936. https://doi.org/10.1038/s41598-023-30563-6.
Dowd, P.F., Naumann, T.A., Johnson, E.T. 2023. Potential role of a maize metallothionein gene in pest resistance. Plant Gene. 34. Article 100409. https://doi.org/10.1016/j.plgene.2023.100409.